Phytochemical profile of Brazilian grapes (Vitis labrusca and hybrids) grown on different rootstocks

Important factors may influence the bioactive compounds in grapes, including scion–rootstock interaction. Therefore, the bioactive compounds and antioxidant activity in grape skin and pulp fractions of ‘Isabel Precoce’, ‘BRS Carmem’, ‘BRS Cora’, ‘BRS Violeta’ and ‘IAC 138–22 Máximo’ were assessed. These cultivars, from genetic improvement programs in Brazil, have good adaptation to subtropical and tropical climate conditions, and can be widely used by winegrowers aiming at adding value to the grape. All grapevines were grafted onto ‘IAC 766’ and ‘IAC 572’ rootstocks under tropical conditions in Brazil. The highest concentration of bioactive compounds was found in skins of ‘BRS Violeta’, followed by ‘IAC 138–22 Máximo’, both grafted onto ‘IAC 766’. There was a strong correlation between phenolic content and antioxidant properties, since antioxidant activity also decreased in the sequence: ‘BRS Violeta’ > ‘IAC 138–22 Máximo’ > ‘BRS Cora’ > ‘BRS Carmem’ > ‘Isabel Precoce’. Skin from hybrid grapes (‘BRS Violeta’, ‘IAC 138–22 Máximo’, ‘BRS Cora’ and ‘BRS Carmem’) grafted in both rootstocks contains higher levels of (poly)phenolic compounds and antioxidant activity than ‘Isabel Precoce’ (V. labrusca). Skin from ‘BRS Violeta’ grafted onto ’IAC 766’ stand out from the others due to their high content of bioactive compounds.


Introduction
Brazilian grape juices are mainly produced from Vitis labrusca grapes, such as 'Isabel', 'Bordô' and 'Concord'. These varieties are well suited to the temperate climate of southern Brazil and experimental area recorded meteorological conditions during the study period. The mean temperature was 24.1˚C, the minimum average was 16.6˚C and the maximum average was 31.7˚C. The average annual rainfall was 1495 mm, with a tendency for concentrated rainfall in the summer months. The soil was classified as 'Argissolo Vermelho-Amarelo' (equivalent to Ultisol, USDA soil taxonomy) according to previously published criteria [23]. The 3-year-old vines were trained on a unilateral cordon system (1 m above the soil) with vertical shoot positioning and spaced 2.0 × 1.1 m apart. Winter pruning was performed in August 2015 and the grapes were harvested in December 2015, when they reached the technological maturation stage [22]. Classical techniques were used to determine the pH, soluble solids and titratable acidity in grape samples. Values ranged from 3.18 to 3.29 for pH, 14.3 to 15.5˚Brix for soluble solids (Brasil, 2002;IN 14, 2018) and from 0.66% to 1.14% tartaric acid for titratable acidity.

Sample preparation and extraction
A sample of 200 berries was randomly collected from the basal, median and apical regions of bunches, divided into four replicates (4 plots, each plot with 50 berries) and fractionated into portions of skin and pulp. The seeds were discarded. The skin and pulp fractions were immediately frozen in liquid nitrogen and stored at −20˚C until analysis.
For the determination of phenolic compounds and antioxidant activity, four plots (each plot with 50 berries) were used for the analyses. Skin and pulp fractions were extracted using > 99% (v/v) ethanol at a sample to solvent ratio of 1 : 2 w/v. The extracts were mixed and incubated for 1 h in a refrigerated shaker (20˚C; Tecnal 1 , Brazil). After centrifugation at 4500 x g for 7 min, 1.5 mL of the supernatant was collected and the residual ethanol was evaporated by a vacuum centrifugal concentrator (miVac DUO concentratior, Genevac 1 , UK). Then, the extract was redissolved (1.5 mL final volume) in acidified water solution (1% phosphoric acid, v/v), filtered through a 0.45 μm membrane (Allcrom 1 , Brazil) and kept frozen at −80˚C until analysis.
From a second sampling, only the pulp portions were used for extraction and analysis of organic acids and individual sugars. In these analyses, other 50 berries were used in each plot (4 plots). The pulp was mixed and diluted in 1 : 1 (v/v) ultrapure water. The extracts were filtered through 0.45 μm nylon membranes (Allcrom 1 , Brazil) and kept frozen at −80˚C until analysis.

Organic acids and sugars determined by HPLC
Organic acids (tartaric, malic, citric, lactic and acetic acids) and individual sugars (glucose and fructose) in pulp were determined using an Agilent 1260 Infinity LC HPLC system (Agilent Technologies, Santa Clara, CA, USA) coupled to a diode array detector (DAD, model G1315D) and refractive index detector (RID, model G1362A), according to the methodology described and validated by Coelho et al. [13]. Separation of the compounds was performed on a Hi-Plex H ion exchange column (300 × 7.7 mm, 8.0 μm internal particles) protected by a PL Hi-Plex H (5 × 3 mm) guard column (Agilent Technologies, Santa Clara, CA, USA). The phase was 0.004 mol L −1 H 2 SO 4 in ultrapure water.

Total phenolic compounds and total monomeric anthocyanins
The total phenolic compound content of the skin and pulp extracts was determined by the Folin-Ciocalteu spectrophotometric method [24]. The absorbance value at 765 nm was compared to a calibration curve and the results were expressed as milligrams of gallic acid equivalent (GAE) per kilogram of skin or pulp (mg kg −1 ). The total monomeric anthocyanin content was determined using the pH-differential method [25] and expressed as malvidin 3,5-diglucoside equivalents in mg L −1 . Both analysis techniques were performed using an UV-Vis spectrophotometer (Instrutherm 1 2000A, Brazil).

Profile of phenolic compounds
The profile of phenolic compounds was determined using a Waters Alliance e2695 liquid chromatograph equipped with a DAD (Waters, Milford, MA, USA) [26]. The column used was a Gemini-NX C18 (150 mm × 4.60 mm, with 3 μm internal particles) and the pre-column was a Gemini-NX C18 (4.0 mm × 3.0 mm), both manufactured by Phenomenex (USA).

In vitro antioxidant activity
The in vitro antioxidant activity was determined using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) [27] and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging methods [28]. For the analysis, the skin and pulp extract samples were diluted with ultrapure water until between 20% and 80% inhibition of the DPPH and ABTS radicals was obtained. The 1 mM DPPH and ABTS radical solutions were prepared in ethanol and diluted to an absorbance of 0.900 ± 0.050 (λ = 734 nm) and 0.700 ± 0.050 (λ = 517 nm), respectively. The absorbances were determined before and after the addition of grape juice using a UV-Vis 2000A spectrophotometer (Instrutherm 1 , São Paulo, SP, Brazil). In the DPPH method, absorbance was measured at times t = 0 and t = 30 min after addition of the sample. In the ABTS method, the absorbance was determined at times t = 0 and t = 6 min after addition of the samples. For both methods, the analytical standard Trolox was used to construct the calibration curves and the results were expressed as equivalents of Trolox per kilogram of grape skin or grape pulp (mM TEAC kg −1 ).

Experimental design and statistical analysis
A completely randomized experimental design was used with ten treatments (scion/rootstock combinations) with four replicates. Data from skin and pulp analysis were submitted to oneway analysis of variance (ANOVA) and means were compared by Tukey test at 5% probability of error, using the statistical program SISVAR version 5.4 [29]. Principal component analysis (PCA) was applied to the 60 variables analyzed in grape skin and pulp, performed through XLSTAT software version 19.4 (Addinsoft, NY, USA).

Organic acids and sugars
In grapes, organic acids and sugars are mainly accumulated in the pulp [10,12]. Tartaric acid was the major compound (4.23 to 6.41 g L −1 ) in most red grapes, except in 'IP-766', which had the highest content of malic acid (7.77 g L −1 ) (Table 1). Therefore, both tartaric and malic acids corresponded to 92.0% and 98.0% of the organic acids quantified. Nevertheless, it has been already confirmed that those acids may account for more than 90.0% of the organic acids found in grapes [10]. The lowest organic acid content was observed in 'CM-572' (7.47 g L −1 ) and the presence of acetic and lactic acids was not detected in any grape berries, which is desirable, since these acids affect stability and characterize microbiological changes in beverages [14].
The highest content of glucose (85.86 g L −1 ) and fructose (77.24 g L −1 ) was observed in 'BRS Carmem'. All grapes showed a glucose/fructose ratio ranging from 1.02 to 1.15, but also a slightly higher glucose-to-fructose-ratio was observed in 'IAC 138-22 Máximo', due to its lower fructose content. Notwithstanding that, these data are close to average (i.e. 1.0) for lateripening grapes [12].
The content and composition of organic acids and soluble sugars determine the organoleptic quality and taste of grapes [10,14], and these characteristics were influenced by the rootstocks. 'BRS Carmem' and 'IAC 138-22 Máximo' grafted on 'IAC 766' rootstock had the highest levels of tartaric and citric acids. In the same rootstock, 'Isabel Precoce' showed a high content of malic acid (7.77 g L −1 ), glucose (79.95 g L −1 ) and fructose (73.83 g L −1 ) ( Table 1). Rootstocks can induce light uptake by grapevine canopies, directly affecting carbon assimilation and storage [17], factors that influence the metabolism of acids and sugars in plants. Variations in the content of malic and tartaric acids, as well as glucose and fructose, were also observed in 'Syrah' grapes grafted on '1103 Paulsen' and '110 Richter' rootstocks, as a function of their difference in vigor [30].

Profile of phenolic compounds
Phenolic compounds were quantified separately in grape skin and pulp extracts from different scion/rootstock combinations. A typical chromatogram of grape skin extracts is shown in Fig 1. Anthocyanin profile. The anthocyanin profile varied significantly according to the scion/ rootstock ( Table 2). The content in grape skins was higher than in pulps. In grape pulp, the anthocyanin content ranged from 0.28 to 11.59 mg kg −1 . The levels of total anthocyanins detected in pulps and skins of 'IAC 138-22 Máximo' and 'BRS Violeta' grapes were the highest compared to the total content of anthocyanins in other grapes. Similar results have been described in 'BRS Violeta' and 'Bordô' (V. labrusca) grape pulps [3,8]. However, the anthocyanin content reached 3025.5 mg kg −1 in VL-766 grape skin, the major anthocyanins in this variety being cyanidin-3,5-diglc (diglucoside), cyanidin-3-glc (glucoside) and delphinidin-3-glc. Such a result was significantly different from those for the other varieties, including VL-572. It is worth noting that the content of malvidin-3,5-diglc in VL-766 (805.3 mg kg −1 ) was higher than that in VL-572 (571.8 mg kg −1 ), evidencing the affinity of 'BRS Violeta' for 'IAC 766'.
Therefore, rootstocks influenced on the anthocyanin content, especially in 'BRS Violeta', in agreement with previous studies that assessed this variety on 'IAC 766' and '106-8 Mgt' rootstocks [31]. Research analyzing the effect of rootstock on hybrid grapes for juice and wine making is scarce, but our results are similar to those from studies on other species that also reported the influence of rootstocks on grape anthocyanin content, i.e. 'Red Alexandria' (V. vinifera) [18] and 'Greco Nero n.' (V. vinifera) [32].
According to Xi et al. [16], in grapes the anthocyanins are synthesized via the flavonoid biosynthetic pathway. However, it is not yet well understood how the rootstock affects the biosynthesis and the content of these compounds. The expression of flavonoid biosynthesis-related genes varies depending on the rootstock [33]. In our study, we also observed variations in the anthocyanin profile according to the rootstock. High concentrations of anthocyanins were found in MX-766 and MX-572 (1640 and 1412 mg kg −1 ) grape skin and more than 50% was represented by malvidin-3,5-diglc, at 878.2 and 738.5 mg kg −1 , respectively. It is worth remembering that malvidin-3,5-diglc was detected at high levels in VL-766 and VL-572. In non-vinifera grape varieties, some studies have reported a large amount of anthocyanins derived from the 3,5-diglucoside group, mainly malvidin, considered markers in these grapes [3,34].
The main anthocyanin found in 'Isabel Precoce' grape skin was malvidin-3-glc (190.6 mg kg −1 ), although it is a V. labrusca variety ( Table 2). This result corroborates those of previous studies on grape juices elaborated from this variety, in which the content of malvidin-3-glc was higher than that of malvidin-3,5-diglc [26,35]. Similar results have also been obtained for grape juices and wines made from 'Isabel' (V. labrusca), which is the most used variety for juice making in Brazil and from which 'Isabel Precoce' originated through a spontaneous somatic mutation [2]. The anthocyanin profile of each variety is commonly linked to its genetic inheritance, although it may be influenced by environmental conditions [3].
VL-766 presented a higher concentration of total monomeric anthocyanins (TMA) than VL-572, i.e. 9.49 and 8.55 g kg −1 of grape skin, respectively. The TMA content was 17 times higher in 'BRS Violeta' than in 'Isabel Precoce' (0.52 mg kg −1 ). Other studies showed low levels of total anthocyanins in grape juices and wines from 'Isabel Precoce' and 'Isabel' [1,26]. Nevertheless, high levels of these compounds have already been observed in 'BRS Violeta' [31,34]. Thus, 'BRS Violeta' becomes an important source of anthocyanins, capable of producing intense color expression in grape juices and young red wines.
Flavonols and trans-resveratrol. The total amount of flavonols was low in 'BRS Carmem', 'BRS Violeta' and 'IAC 138-22 Máximo' grape pulps, as values ranged from 0.04 to 0.53 mg kg −1 ; none were detected in 'Isabel Precoce' or 'BRS Cora' ( Table 2). A low flavonol content was also found in 'Bordô' grape pulp (1.44 μmol kg −1 ), about 100 times less than in grape skin [8]. The flavonol content ranged from 7.83 to 61.89 mg kg −1 in grape skins and isoquercetin was the major phenolic compound in all samples of grapes, being responsible for more than 50% of the total flavonol content in most samples. The highest isoquercetin level was found in MX-572, MX-766 and VL-766.
Phenolic acids and flavanols. The phenolic acid content in grape skin was higher than in pulps and ranged from 5.07 to 35.31 mg kg −1 . This result corroborates those of the studies conducted by Rebello et al. [34] and Lago-Vanzela et al. [8], who also observed a greater distribution of phenolic acids, especially hydroxycinnamic acids, in skins than in pulps of 'BRS Violeta' and 'Bordô' grapes, respectively. The highest levels of phenolic acids were obtained in 'BRS Violeta' and 'Isabel Precoce', with an average of 33.06 and 20.45 mg kg −1 , respectively, followed by 'IAC 138-22 Máximo', 'BRS Cora' and 'BRS Carmem' (13.65, 12.51 and 5.22 mg kg −1 , respectively). Phenolic acid was also observed, in 'BRS Violeta' > 'Isabel Precoce' > 'BRS Cora' grape juices from the São Francisco Valley (Northeast, Brazil) [26]. Phenolic acids were genotype-dependent, as were the other phenolic compounds. In grape skin, the major phenolic acid was gallic acid in 'BRS Carmem', cinnamic acid in 'BRS Violeta' and caffeic acid in 'Isabel Precoce', 'BRS Cora' and 'IAC 138-22 Máximo'. In all grape pulps, gallic acid was the most abundant phenolic acid. Gallic acid is described as one of the most important phenolic compounds in grapes, as it is the precursor of all hydrolyzable and condensed tannins, that is, compounds of sensory interest [36].
The flavanol content was similar between cultivars, values ranging from 0.26 to 0.72 mg kg −1 in grape pulps and from 1.98 to 6.19 mg kg −1 in grape skins ( Table 2). The latter grape fraction presented a high epicatechin gallate content in 'BRS Violeta', while the highest epigallocatechin gallate content was found in 'BRS Carmem'. There was no effect of the rootstocks in each grape variety. Flavanols are tannins that are mainly located in grape skin and seeds [19], which explains the low values found in pulp. The amount of flavonols in 'Greco Nero n.' has been shown to be little influenced by rootstock, suggesting a strong genotypic influence of the scion on the accumulation of these compounds in grape skin [32].

PLOS ONE
Phytochemical profile of Brazilian grapes   The authors found the highest phenolic content in these varieties in relation to other vinifera and non-vinifera grape varieties. Grape skin had predictably higher antioxidant activity (AOX) than pulp, due to the higher content of phenolic compounds (Fig 2). In grape pulp, AOX ranged from 0.73 to 1.37 mM TEAC kg −1 for the DPPH method, and from 0.20 to 1.02 mM TEAC kg −1 for the ABTS method ( Fig 2B). For both methods, high values were found in VL-766, differing significantly from VL-572. The effect of rootstock on the AOX of 'BRS Violeta' is related to the high phenolic content provided by 'IAC 766' rootstock, indicating the influence of rootstock on the phenolic compound content, as observed by Cheng et al. [18] in 'Red Alexandria' grape pulp.
Although the results for phenolic compounds showed an effect of rootstock on grape skins, mainly on 'BRS Violeta', there was no influence of 'IAC 766' and 'IAC 572' rootstock on the AOX of grape skin, which ranged from 7.20 to 30.79 mM TEAC kg −1 for the DPPH method and from 7.33 to 85.05 mM TEAC kg −1 for the ABTS method. In grape skins, the highest AOX (DPPH and ABTS) was observed in 'BRS Violeta' (30.9 and 83.9 mM TEAC kg −1 ), followed by IAC 138-22 'Máximo' (24.1 and 40.4 mM TEAC kg −1 ) (Fig 2A). The lowest AOX was found in 'Isabel Precoce', 7.4 and 7.8 mM TEAC kg −1 using the DPPH and ABTS methods, respectively. The variation between grape varieties is related to the phenolic content, indicating that grapes rich in phenolic compounds also have higher AOX. Other studies have demonstrate these results not only in grapes, but in juices and wines [37,38].

Principal component analysis
Aiming at a descriptive model for grouping organic acids, sugars, phenolic compounds and AOX with scion/rootstock combinations, the results were compared using PCA. PC1 and PC2 explained 70.12% of the data variance (Fig 3). VL-766 and VL-572 grapes were grouped in PC1+ and PC2− ( Fig 3A) and this result may be attributed to the phenolic compounds, mainly cyanidin-3,5-diglc, delfinidin-3-glc, cyanidin-3-glc, pelargonidin-3-glc, isorhamnetin, resveratrol and (−)-epicatechin gallate, besides TPC and AOX (DPPH and ABTS), related to grape   Fig 3B). In addition, other compounds such as myricetin, chlorogenic acid, ρ-coumaric acid, cinnamic acid, gallic acid and TMA found in grape skins, and isoquercetin and kaempferol from grape pulps contributed to the grouping in PC1+. It is worth mentioning that despite both being grouped in PC1+ and PC2−, the highest levels of these compounds were detected in VL-766, demonstrating the possible influence of 'IAC 766' rootstock on the antioxidant composition of grapes ( Fig 3B and Table 2).
'Isabel Precoce', 'BRS Cora' and 'BRS Carmem' were grouped in PC1− and PC2− (Fig 3A), regardless of the rootstock. In 'BRS Carmem' pulp the highest levels of glucose and fructose were observed (Fig 3B), results that may have contributed to the grouping of these grapes in PC1− and PC2−.

Conclusions
In this study, there were differences in the phytochemical profile of grapes grown on different rootstocks. Although organic acids were evenly distributed among the evaluated grapes, 'BRS Carmem' had the highest sugar levels. The content of phenolic compounds in the grape skin was higher than in the pulp. Overall, 'BRS Violeta' surpassed the others, since it presented the highest concentrations of all studied (poly)phenol compounds. AOX decreased in the following order: 'BRS Violeta' > 'IAC 138-22 Máximo' > 'BRS Cora' > 'BRS Carmem' > 'Isabel Precoce'. Despite the rootstocks having little influence on the evaluated compounds, their effect was relevant to 'BRS Violeta' grafted on 'IAC 766'. Finally, the phytochemical profile of grapes ranged according to the variety and rootstock used in cultivation and presents the characterization of Brazilian grape varieties grown on Brazilian rootstocks.